Immune hyporeactivity to bacteria and multiple TLR-ligands, yet no response to checkpoint inhibition in patients just after meeting Sepsis-3 criteria

Rationale The immune profile of sepsis patients is incompletely understood and hyperinflammation and hypoinflammation may occur concurrently or sequentially. Immune checkpoint inhibition (ICI) may counter hypoinflammation but effects are uncertain. We tested the reactivity of septic whole blood to bacteria, Toll-like receptor (TLR) ligands and to ICI. Methods Whole blood assays of 61 patients’ samples within 24h of meeting sepsis-3 criteria and 12 age and sex-matched healthy volunteers. Measurements included pattern/danger-associated molecular pattern (P/DAMP), cytokine concentrations at baseline and in response to TLR 2, 4, and 7/8 ligands, heat-inactivated Staphylococcus aureus or Escherichia coli, E.coli lipopolysaccharide (LPS), concentration of soluble and cellular immune checkpoint molecules, and cytokine concentrations in response to ICI directed against programmed-death receptor 1 (PD1), PD1-ligand 1, or cytotoxic T-lymphocyte antigen 4, both in the absence and presence of LPS. Main results In sepsis, concentrations of P/DAMPs and inflammatory cytokines were increased and the latter increased further upon incubation ex vivo. However, cytokine responses to TLR 2, 4, and 7/8 ligands, heat-inactivated S. aureus or E. coli, and E. coli LPS were all depressed. Depression of the response to LPS was associated with increased in-hospital mortality. Despite increased PD-1 expression on monocytes and T-cells, and monocyte CTLA-4 expression, however, addition of corresponding checkpoint inhibitors to assays failed to increase inflammatory cytokine concentrations in the absence and presence of LPS. Conclusion Patients first meeting Sepsis-3 criteria reveal 1) depressed responses to multiple TLR-ligands, bacteria, and bacterial LPS, despite concomitant inflammation, but 2) no response to immune checkpoint inhibition.

hours, those under 18 years of age, on immunosuppressive medications (taking over 30 mg/d cortisol equivalent), or with HIV infection were excluded. Blood from 61 patients that had been drawn via central catheters into tubes containing 16 IU/ml unfractionated heparin (02.1064, Sarstedt, Nümbrecht, Germany) was available and transferred to our laboratories at room temperature within 20-30 minutes. Medical history, clinical status, medication, hemodynamic, hematological, and laboratory data (Table 1), results from cultures of blood and other sources, and PCR (SeptiFast, Roche, Basel, Switzerland) for DNA typical for 20 frequent pathogens (S1 Table) were documented. SAPS-II and SOFA-Scores were calculated, and patients assessed for the presence of septic shock, positive qSOFA, and SIRS criteria (Table 1). Twelve healthy adults served as age and sex matched controls following ethics committee approval (#17-7869-B0) and informed consent. Patient care and laboratory personal were unaware of all (anonymized) data to avoid bias. Since neither patients nor volunteers underwent health related interventions registration as a clinical study was not applicable, as confirmed before enrollment by the German clinical study register.

Whole blood incubation
Twohundred μl of heparinized whole blood were pipetted to wells of 96-well polystyrene plates (Nunc 262162, Thermo Fisher Scientific, Waltham, MA). Subsequently, inhibitors or stimulants were added, and suspensions incubated for 6 or 22 hours under standard conditions (37˚C, humidified atmosphere, 5% CO 2 ) upon which supernatant mediator concentrations were measured. Individual assays were run in triplicate and pooled for luminex ELISA (see below).

Mediator and soluble checkpoint molecule concentrations in blood plasma and blood cell incubation supernatants
Concentrations of proteinaceous inflammatory mediators in blood plasma (before or after whole blood incubation) were measured by multiplex ELISA (Luminex LXSAH-06, R&D Systems, Minneapolis, MN) against a standard curve according to the manufacturer's instructions. Standards were included in the sets. We chose four cytokines commonly associated with human sepsis (TNF, IL-6, IL-1β and α), IL-8 as major granulocyte attractant, and IL-10 as an important anti-inflammatory cytokine.

Expression of cellular checkpoint molecules
Expression of cellular checkpoint molecules was assessed by flow cytometry. Leukocytes from blood were isolated using an aggregation and gravitational separation method (HetaSep TM , STEMCELL TM technologies, Vancouver, Canada) according to the manufacturer's instructions and stored in BamBanker freezing medium (STEMCELL TM technologies). About 5 x10 6 leukocytes were later stained on ice for 30 min (staining buffer, BioLegend, San Diego, CA). After washing, cells were taken up in 50 μl staining buffer containing a dead cell detection reagent (Zombie aqua TM , BioLegend). Expressions were measured by flow cytometry (Cytoflex s, Beckman Coulter, Brea, CA) and analyzed using a DeNovo software (FCS Express 7, Pasadena, CA). Details on antibodies used, FACS-settings, and gating strategy are given in the S2 File.

Quantification of circulating host mitochondrial, genomic, and bacterial DNA in blood plasma
Total DNA was isolated from 200 μl of plasma (QIAamp DNA Blood Mini Kit, Qiagen, Venlo, The Netherlands) to serve as template for all real-time quantitative PCRs performed by applying a StepOne Plus PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA). A melting curve was performed after each run.
Human nuclear DNA was quantified by application of the forensic grade human genomic DNA quantification assay (gDNA detection kit, Primerdesign Ltd., Chandler's Ford, UK) and the Taqman Fast Advanced Master Mix (Applied Biosystems, Thermo Fisher Scientific). Briefly, amplification of a single copy region of non-transcribed DNA was performed with provided primers and a hybridization probe. A standard curve was generated upon serial dilution of a provided positive control template in parallel to each run. Cycling conditions were 2 min at 50˚C, 20 s at 95˚C, followed by 50 cycles of 7 s at 95˚C and 40 s at 60˚C.
Bacterial DNA was quantified by application of Eubacteria genesig1 Standard Kit and the oasig lyophilised 2x qPCR Master Mix (Primerdesign Ltd.) according to the manufacturer's instructions to amplify a specific 16S ribosomal RNA gene segment. A standard curve was prepared by serially diluting the Eubacteria positive control template in each run. Cycling conditions were 2 min at 95˚C, 50 cycles of 10 s at 95˚C and 60 s at 60˚C.

Analysis of sepsis patients' and control blood plasma samples for their propensity to activate ectopically overexpressed TLR2 read out as NF-κB dependent luciferase reporter gene activation
Luciferase assaying for analysis of TLR2 ligand activity has been described earlier [3]. In short, TLR2 expression and luciferase reporter plasmids were transiently transfected into largely TLR deficient human embryonic kidney fibroblastoid HEK293 cells to assess TLR2 specific P/ DAMP driven NF-κB activation [17]. Twenty μl of blood plasma in 80 ml RMPI 1640 was added to individual wells of 96 well cell culture plates, which were incubated at standard incubation conditions for 16 hours upon which cells were lysed. Reporting and control luciferase activities in cellular lysates were measured with a 96-well plate luminescence reader (Orion II, Titertek-Berthold, Bad Wildbad, Germany).
Immune checkpoint inhibition (ICI) in sepsis patients' whole blood samples in the absence or presence of E. coli lipopolysaccharide. ICI can unleash activity of cells inhibited by checkpoint molecules. Antibodies against PD-1 (Nivolumab, Bristol-Myers Squibb, New York City, NY, or Pembrolizumab, MSD, Kenilworth, NJ), PD-L1 (Atezolizumab, Genentech, San Francisco, CA), or CTLA-4 (Ipilimumab, Bristol-Myers Squibb) were added at the start of whole blood sample incubation to yield a final concentration of 30 μg/ml (corresponding to those in anti-cancer therapy [16]) and assays incubated for 22 hours. Supernatant mediator concentrations were measured subsequently in the absence and in the presence of E. coli LPS (see above) added 6 hours after incubation start in concentrations of 1 ng/ml or 10 ng/ml. Continuous clinical variables are presented as means ± standard deviation (SD), discrete clinical variables as percentage of the respective cohort or median with quartiles, clinical data were descriptive. Data were analyzed for non-normality using the Anderson-Darling test. Non-normally distributed values are presented as dot plots with medians and quartiles. All hypotheses were set a priori, and statistical tests were two-tailed. We used the Mann-Whitney U test for unpaired and the Wilcoxon matched pairs signed rank test, as well as the Friedman test with Dunn's multiple comparison corrected post-test for paired data, as appropriate. Pvalues were corrected for multiple testing using the Holm-Bonferroni procedure with increase of the p-values (instead of lowering the local α-level), and p-values <0.05 after correction were considered statistically significant. The available sample volume limited the number of intraassay interventions per patient so that not all types of assay interventions/measurements could be performed on samples from all patients. Twelve healthy volunteers where enrolled over the whole study period. As available and deemed appropriate, 4 to 11 control samples per experiment were run in parallel with sepsis patients' samples.

Statistical methods
To screen for variables potentially predicting in-hospital-mortality we initially compared all ex vivo data between hospital survivors and hospital non-survivors and tested for significant differences via the Mann-Whitney test. Of these, IL8 at baseline and IL6 after 6 hours of LPS stimulation (IL8[baseline] and IL6[LPS]) significantly differed between hospital-survivors and hospital non-survivors: We then performed a multiple logistic regression (with Graph Pad Prism standard settings) with in-hospital survival as dependent variable and the significant variables of the first step as independent variables. As IL8 at baseline and IL6 after 6 hours of LPS stimulation significantly correlated (p = 0.003, Spearman's rank correlation), the assumption of independence was violated. We hence eliminated one variable previously assumed to be independent to choose the model with the lower Akaike information criterion corrected for small sample sizes (AICc

Results
Please refer to S1 Fig for a concise summary of findings.

Characteristics of sepsis patients and controls
As expected, inflammatory, hemodynamic, and metabolic markers were abnormal in patients (Table 1) just meeting Sepsis-3 criteria. About a third of sepsis patients' blood cultures and a sixth of microbiological diagnostic germ DNA PCRs were positive, and pneumonia was the most common focus (S1 Table). Patients were rather sick with a fatality rate of approximately 50% (Table 1). While patients' and volunteers' leukocyte concentrations differed, their monocyte concentrations as the major source of inflammatory cytokines did not (Table 1).

Depressed inflammatory cytokine response to TLR2, 4, and 7/8 ligands
Supernatant inflammatory cytokine concentrations upon incubation with TLR 2, 4, and 7/8 ligands increased in samples from both sepsis patients and healthy volunteers compared to vehicle (Fig 1). However, when compared to the concentrations seen in stimulated samples from healthy subjects, this increase was markedly less, although concentrations in sepsis patients at baseline were greater (Fig 1, Table 1). Thus, inflammatory cytokine concentrations in response to multiple TLR-agonists were significantly depressed in assays of sepsis patients compared to healthy volunteers. This applied to IL-6 for the TLR2 ligand (median quotient: 2.6-fold, p = 0.0022), TLR4 ligand (6,8-fold, p<0.0004), and the TLR7/8 ligand (1.05-fold, p = 0.0073) as well as to TNF (5.5-30.6-fold) and IL-1β (11-25-fold) for these ligands (Fig 1).
In contrast, the concentration of IL-10 in sepsis patients significantly increased even beyond values seen in samples from healthy subjects following stimulation with the TLR4 ligand (p = 0.0036), but not significantly following stimulation with TLR2 or 7/8 ligands.

Depressed cytokine responses to Gram-positive and Gram-negative bacteria in assays of sepsis patients
Cytokine concentrations attained in response to S. aureus as well as to E. coli were markedly and significantly less in assays of sepsis patients than in those of healthy controls, as observed for IL-6 (median ratio: 3.3 & 2.1-fold), TNF (24 & 92-fold), IL1-α (15.8 & 18-fold), and IL-1β (132 & 527-fold), respectively, whereas no differences were seen for IL-10 and IL-8 (Fig 2). Taken together, inflammatory cytokine concentrations evoked in assays were depressed both in response to multiple TLR-ligands and inactivated bacteria in samples from sepsis patients when just meeting Sepsis-3 criteria.

Depressed cytokine response to an E. coli LPS challenge associated to mortality
Supernatant inflammatory cytokine concentrations after 6 hours of whole blood incubation with E. coli LPS were markedly depressed in assays of septic patients compared to healthy controls when incubated for the same time ( Fig 3A). This was seen for IL-6 (7.1-fold, p<0.0006), TNF (20-fold, p = 0.0006), IL-1α (18.6-fold), and IL-1β (21.5-fold, both p<0.0006). In contrast, anti-inflammatory IL-10 increased upon incubation when compared to samples from controls.
Stratifying the changes in IL-6 evoked by LPS in assays from sepsis patients there was a significant association of decreased IL-6 concentrations with in-hospital mortality (p = 0.046, Fig  3B). The receiver-operator-characteristic (ROC; Fig 3C) revealed an AUC of 0.7 for prediction of in-hospital mortality (95%-CI: 0.55-0.86; likelihood ratio test: p = 0.0137).

PLOS ONE
Immune hyporeactivity and no response to immune checkpoint inhibition in early sepsis Circulating P/DAMPs and inflammatory cytokine production in whole blood assays mtDNA concentrations (ATPase-6 and D-loop) were markedly increased (p<0.0001 each) in samples from sepsis patients as were concentrations of nuclear (host genomic) DNA (p = 0.0001), and, to a lesser extent, bacterial DNA (p = 0.032, Fig 4A). As exemplified by a TLR-2 and NF-κB dependent luciferase assay (Fig 4B, p = 0.0004) and greater baseline cytokine concentrations (Table 1), inflammatory activity was increased in plain samples from sepsis patients compared to controls. Supernatant concentrations of the inflammatory cytokines IL-6 and TNF, even without additional stimuli, further increased over the 6 hour span of incubation in samples from septic patients but not in controls (Fig 4C), suggesting ongoing cytokine production ex vivo.

Expression of cellular and soluble checkpoint molecules and the impact of checkpoint inhibition on cytokine concentrations
The percentage of T-cells and monocytes expressing PD-1 was significantly increased in samples from sepsis patients relative to healthy age-matched controls, whereas soluble PD-1 concentrations were not changed (Fig 5). CTLA-4 expression on monocytes was also increased When samples were incubated with monoclonal antibody (mAB) checkpoint inhibitors directed at PD-1, PD-L1, and CTLA-4, respectively, there was no relevant or statistically significant increase in cytokine concentrations after 22 hours of incubation relative to controls, both in the absence and presence of 1 or 10 ng/ml LPS (Fig 6).

Discussion
The present study, to our knowledge, is the first to provide a comprehensive view of bloodborne immunologic features of patients just meeting the hallmarks of sepsis, as defined by the Sepsis-3 criteria. While previous publications on septic immunosuppression mostly focused on late sepsis [22,23] or amalgamated LPS-tolerance with broader septic immune tolerance [23], we observed a markedly mitigated cytokine production in response to multiple TLRligands (2, 4, and 7/8), to both Gram-positive S. aureus and Gram-negative E. coli, and to an E. coli LPS challenge within 24 hours of sepsis onset. At the same time, the hosts'blood revealed increased inflammatory cytokine concentrations further increasing on incubation and increased P/DAMP concentrations. Thus, functions of the innate immune system are depressed already very early in sepsis and this hyporeactivity occurs concurrently with inflammatory sepsis features, i.e., clinical symptoms and signs, and inflammatory cytokine production. With few exceptions [6], most previous work has described septic hyperinflammation and hypoinflammation as distinct and subsequent phases [1,22]. Collecting data from different experimental setups, concomitant hyper-and hypoinflammation in sepsis has been discussed [13]. This study, however, demonstrates effects of both these elements of sepsis pathology at the same time and within the same model system. That this has a clinical impact is suggested by an increased mortality in patients with a lesser in vitro cytokine response to LPS. Despite such hyporeactivity and increased checkpoint molecule expression, however, immune checkpoint inhibition did not spark cytokine production ex vivo both in the absence or presence of LPS.
Although the understanding of sepsis mechanisms and supportive therapy have improved mortality, causal therapies appear not in sight [1,24], possibly because translation from animal experiments is limited [8,9,25,26] and it is difficult in practice to obtain and process samples in a timely fashion. While previous definitions of sepsis had been somewhat blurry the more recent Sepsis-3 definition has provided a common time anchor for the diagnosis of sepsis. Accordingly, we analyzed sepsis associated blood-borne immune responses with respect to a specific time, i.e., within 24 hours of first meeting Sepsis-3 criteria but not later. Rather than isolated cells we used the patients'whole blood that should incorporate all principle components of blood related inflammation, such as all leukocytes, causative pathogens, if still present, and the assortment of P/DAMPs and mediators prevailing in hosts which are known to stimulate TLRs [27,28]. We chose this setup over cell isolation to model septic inflammation with as  little manipulation as possible and including the complex interplay between all blood-borne cell types, their natural medium (plasma) and host plasma components [27]. While such systems might be preferable to cellular isolation when describing the net effect of complex systems or interventions, additional mechanistic insight might have been gained from repeating the experiments in more reduced model systems missing host plasma and other cell components, but this was beyond the scope of this study as sample volume available from critically ill patients limited the number of experiments we could perform per sample.
We found inflammatory supernatant cytokine concentrations of sepsis patients' specimens to further increase ex vivo. This was likely mediated by increased concentrations of several P/ DAMPs in the hosts'autologous plasma such as mitochondrial, genomic, and bacterial DNA, which were all markedly increased compared to controls, as exemplified by increased TLR2 agonistic activity. However, a maintained cellular program, previously activated in vivo, may have contributed. To our knowledge, such an ongoing ex vivo inflammation of whole blood has not been demonstrated before and might be an interesting model system for further studies on manipulating septic hyperinflammation. Furthermore, this set-up might in future studies allow a follow-up over the later course of sepsis.
At the time of this inflammatory response, however, inflammatory cytokine concentrations evoked in response to TLR2, TLR4, and TLR7/8 ligands were all markedly depressed compared to controls. This argues for a decrease in TLR receptor availability on cell surfaces, an inhibited TLR transduction process, and/or a decreased cytokine output due to other intracellular mechanisms, i.e., negative intracellular regulators [29], epigenetic regulation, or miRNAs. That cell function was not impaired by the assays themselves was confirmed by a spiking increase in cytokines after 6 hours of incubation when E. coli LPS was added to the assays. While we did not explore the molecular mechanisms contributing to this decreased reactivity to several TLR-ligands, depressed reactivity to ligands of multiple receptors of the TLR-family early in sepsis is interesting and may mechanistically relate to LPS tolerance [30][31][32].
Inflammatory cytokine responses to Gram-positive and Gram-negative bacteria were markedly depressed as well, possibly by the same mechanisms. This implies a broad inflammatory hyporesponsiveness reaching beyond individual signaling axes. Specifically, evoked supernatant concentrations of TNF, IL-6, IL-1α, IL-1β, and IL-8 were all substantially less in assays of sepsis patients' samples, despite greater baseline concentrations and ongoing inflammatory activity when compared to controls. This was true both for S. aureus and E. coli which are common and prototypical strains related to sepsis [33]. Together, this indicates that at least some mechanisms in the defense against bacteria, i.e., cytokine secretion in response to bacterial encounters, are impaired very early in sepsis, i.e., already during the first day of sepsis.
The inflammatory cytokine response to an E. coli LPS challenge ex vivo was also much less in blood from sepsis patients than in control assays and this depressed LPS-evoked IL-6 production was associated with increased in-hospital mortality. A depressed cytokine response to LPS had previously been suggested as a potentially useful tool for monitoring in a report of 10 mixed surgical patients with sepsis of various duration [34].
Together, these data indicate an immune hyporeactivity present already within the first 24 hours of meeting Sepsis-3 criteria and occurring concurrently with inflammation. This strongly contradicts the canonical model of sepsis as an early hyperinflammation followed only later by hypoinflammation and immunosuppression, as proposed by the CARS concept [11]. A recent review updating present views of sepsis argued that a phase of "hyperinflammation" with organ dysfunction is followed later either by recovery, persistent inflammation, or immunosuppression and nosocomial infection, catabolism, and often death [22]. Our data indicate that at least some features of immunosuppression, notably the markedly decreased inflammatory cytokine secretion in response to multiple TLR-ligands and to bacteria occur already very early in sepsis (i.e., when just meeting Sepsis-3 criteria), concurrently with inflammation, and thus reveal a Janus-faced immune profile at this time. Such DAMP evoked reprogramming may also explain why patients with abacterial insults such as trauma, stroke, burns, or major surgery are prone to early infection [35][36][37].
Other reports have suggested early septic immunosuppression of blood cells [6] and splenocytes [5], although interpretation was hampered by concurrent corticoid treatment, use of isolated antigens, a different or wide-ranged timing of taking samples, and/or different sepsis inclusion criteria. Still others have cautioned that rather than designating such findings "immunosuppression", or "immunoparalysis" one should more neutrally use the term "immune reprogramming" [13,14] to account for potentially different mechanisms on the tissue level.
Regardless of terminology, sepsis patients are believed to enter an immune state fostering late infections [38][39][40]. Since one proposed mechanism contributing to septic "immunoparalysis" [4] is immune checkpoint molecule expression [41] this raised the question whether in sepsis immune checkpoint inhibition can unleash host defenses previously inhibited via immune checkpoint molecules [42], so as to increase immune activity and improve outcome. Cellular expression of CTLA-4, PD-1, or PD-L1 [43,44] was increased in our patient cohort and we targeted these molecules to assess whether the cytokine response would increase. However, inflammatory cytokine concentrations did not change compared to controls with any of these checkpoint molecule antibodies, even when applied in concentrations similar to cancer therapy, and neither in the absence nor in the presence of different LPS doses as a second stimulus. Clinically, this finding is disappointing, in particular since we had found significant increases in concentrations of several checkpoint molecules in the septic patients'blood, as have others [45][46][47][48][49]. As checkpoint inhibition is considered a promising therapeutic modality in sepsis to counter immunosuppression and prevent nosocomial infections [50], this study adds relevant preclinical data regarding its potential effect. Whether these negative results with checkpoint inhibition correspond to a particular immune status, mirror unknown clinical features, or reflect immunosuppression mediated by yet untested checkpoint molecules remains unclear. Despite some in vitro and animal studies it remains unresolved whether ICI is effective in sepsis models or not [51][52][53]. Regarding their use in human sepsis, to our knowledge, no randomized controlled trial has been made beyond assessment of safety, tolerability, and pharmacokinetics [54,55].
Since the early septic immune hyporesponsiveness appears to be unresponsive to PD1/L1 or CTLA4 inhibition, however, our findings question the potential use of checkpoint inhibitory treatments during early sepsis. Nevertheless, as the immune status may change during the further course of sepsis, timing or patient selection may be key and it remains to be assessed whether such approaches might be promising at later time points, only in specific patients, or under different circumstances. Furthermore, it is possible that the observed hyporeactivity might be alleviated by peptide receptor stimulation [56] rather than checkpoint blockade.
Sepsis is a heterogenous disease with diverse etiologies and host responses that in individuals may prevail to different degrees at different times. Accordingly, we strictly adhered to the recent Sepsis-3 criteria [10] and enrollment and taking samples within 24 hours. Nevertheless, despite using these criteria our data reveal some dispersion of clinical disease severity, P/ DAMP concentrations, and checkpoint molecule expressions. Thus, while Sepsis-3 criteria are considered a step forward to unify recognition, definition of diagnosis, and epidemiology of sepsis, it appears less certain whether this definition helps to provide a common time anchor to fully understand prevailing mechanisms. Thus, assessing immune phenotypes prevailing in individual sepsis patients along their course may help to better understand these mechanisms and assays such as those used in this study may provide a toolbox to gain further insights.

Limitations
Obviously, whole blood incubation ex vivo, while perhaps preferable to more reduced models, does not consider the interplay between blood and other body compartments or organs. Thus, our conclusions are limited to blood borne immunity. However, immunosuppression has been reported after a median of 8 days, albeit with a range of 1-195 days [5], in splenocytes of ICU patients dying from sepsis and multiple organ failure, suggesting that hyperinflammation and "immunosuppression" may or may not have a mutual time delay on the tissue level. While our clinical therapy followed established guidelines [57] and we did not apply high-dose corticoid therapy, we cannot rule out that other drugs may represent hidden confounders versus "natural" untreated sepsis which to study is not feasible ethically. Furthermore, as a tertiary referral center, we cannot exclude selection bias since our patients, although first meeting sepsis-3 criteria, represent a severely ill cohort and different results may have been observed with less severe sepsis.

Conclusion
Within 24 hours of meeting sepsis-3 criteria, there is a markedly mitigated inflammatory cytokine production ex vivo in response to TLR2, 4, and 7/8 ligands as well as to bacteria prototypical for evoking sepsis such as Gram-positive S. aureus and Gram-negative E. coli. Thus, the immune system is depressed very early in sepsis and already at the same time as inflammatory key features of sepsis prevail. This Janus-faced profile contradicts the canonical late timing of immunosuppression and supports the presence of early immunosuppression already when patients are just meeting sepsis-3 criteria; yet the missing cytokine response to ICI despite increased expression of the corresponding checkpoint molecules appears to advice against immune checkpoint inhibition at this time point.